Method of isolating ependymal neural stem cells

ABSTRACT

The present invention relates to a method of isolating ependymal neural CNS stem cells from a post-natal animal or a human, which method comprises the steps of  
     (a) screening single cells obtained by dissociating CNS tissue from said animal for cells exhibiting at least one characteristic of an ependymal neural stem cell; and  
     (b) recovering the cells that exhibit the characteristic or characteristics screened for in step (a).  
     The screening may be performed for a specific cell surface protein or by previously labeling the ependymal cells.  
     The invention also relates to isolated ependymal neural CNS stem cells, in vitro and in vivo assays based on the findings according to the invention and various uses of the ependymal neural stem cells according to the invention.

TECHNICAL FIELD

[0001] The present invention relates to a method of isolating cells thathave never before been identified and located from the mammaliancentral-nervous system. The invention also relates to such isolatedcells per se and to various uses thereof as well as assays using thesame.

BACKGROUND

[0002] Until recent years, a ‘static’ view on the fate of nerve cells inthe central nervous system (CNS) was universally prevailing, based onthe assumption that new neurons could not be generated in the adultmammalian brain. However, such renewal of neurons has been described incertain regions of the adult CNS, e.g. in the olfactory bulb, wheresignals from neurons from the organ of smell reach the brain (Kaplan etal., Science 197:1092) and in the dentate gyrus of hippocampus (Bayer etal., Science 216:890). Since neurons are unable to divide, the additionof new neurons suggested the existence of immature cells, i.e.progenitor or stem cells, which may generate neurons. Evidencesupporting the existence of a multipotent neural stem cell in the adultmammalian CNS was presented a few years ago (Reynolds et al., Science255:1707). However, as in several other organs, the realization of theexistence of a stem cell has come before identifying and localizing thesame. Interestingly, neurogenesis in the adult brain persists throughoutadulthood in rodents (Kuhn P G, J. Neurosci. 16:20) and seems to be anevolutionary well conserved phenomenon present in a variety of mammals(Gould et al. J. Neurosci. 17:2492, Gould et al., Proc. Natl. Acad. Sci.USA 95:3168). In humans, the issue is difficult to address, althoughexperimental data from cultures of adult human brain tissue(Kirschenbaum et al, Cereb. Cortex 6:576) suggest that there may becontinuous neurogenesis also in the adult human CNS.

[0003] The existence of neural stem cells in the adult mammalian CNS wasfirst demonstrated by culturing cells from the adult rat brain andspinal cord. Under certain culture conditions a population ofmultipotent neural stem cells can be propagated from dissociated adultrat brain and spinal cord (Reynolds et al., Science 255:1707, Dev. Biol.175:1, Weiss et al., J. Neurosci. 16: 7599). The culture medium has tocontain a mitogenic factor, e.g. epidermal growth factor (EGF) orfibroblast growth factor (FGF), and serum must be excluded. In contrastto stem cells, most other CNS cell types do not survive in thesecultures.

[0004] Under these conditions, single cells proliferate in vitro and theprogeny forms a cluster of aggregated cells (Reynolds et al., Science255:1707, Dev. Biol. 175:1). Such cell clones detach from the culturedish after a few days in vitro. The cells continue to proliferate andform a characteristic spheroid cell aggregate, referred to as aneurosphere, of tightly clustered cells, all of which are derived from asingle cell. Most of the cells in the neurosphere express nestin, anintermediate filament found in neuroepithelial stem cells. (Lendahl etal., Cell, 60:585), but not markers typical for differentiated cells.These undifferentiated cells rapidly differentiate if plated on anadhesive substrate or if serum is added to the culture medium.Importantly, a clone of cells derived from a single cell can generateneurons, astrocytes and oligodendrocytes, demonstrating that at leastthe initial cell was multipotent (Reynolds et al., Science 255:1707,ibid. Dev. Biol. 175:1). Moreover, if a cell clone is dissociated, manyof the cells will form new clusters of undifferentiated multipotentcells (Reynolds et al., Dev. Biol. 175:1), thus fulfilling the criteriafor being stem cells.

[0005] Thus, the method above suffers from the serious drawback that thecell population used is of a complex, mixed composition. Even though ithas been possible to enhance the growth of some cell types, it isimpossible to draw any conclusions regarding the original localizationof the cells obtained.

[0006] Consequently, other methods have been proposed to determine thelocalization of the adult CNS stem cells, wherein different parts of theadult rodent forebrain have been carefully dissected and cultured totest for the capacity of neurogenesis. These studies have demonstratedthat stem cells are most abundant in the wall of the lateral ventricleand in the hippocampus (Lois et al., Proc. Natl. Acad. Sci. USA,90:2074, Morsehead et al., Neuron 13:1071, Palmer et al., Mol. Cell.Neurosci. 6:474, ibid, 8:389). Furthermore, stem cells can be isolatedfrom the walls of the third and fourth ventricles as well as from theadult spinal cord, suggesting the presence of stem cells adjacent to theventricular system along the entire neuraxis (Weiss et al., J. Neurosci.16: 7599).

[0007] However, the exact localization and identity of the neural stemcell has been enigmatic. The wall of the lateral ventricles has been thesubject of detailed morphological studies (Doetsch et al., J. Neurosci.17:5046). The ventricular system is lined by a single layer of ependymalcells. Mammalian ependymal cells have traditionally been considered tobe highly specialized cells with the main function to form a barrierbetween the nervous tissue and the cerebrospinal fluid (Del Bigio, Glia14:1), which strongly argues against these cells being undifferentiatedstem cells. Beneath the ependymal layer is the subependymal layer, alsoknown as the subventricular zone. This area harbors astrocytes,neuroblasts and progenitor cells (Doetsch et al., J. Neurosci. 17:5046).The progenitor cells in the subependymal layer have a high proliferationrate (Morsehead et al., J. Neurosci. 12:249). Generally, stem cellsproliferate very slowly and when the rapidly proliferating subependymalcells were selectively killed, the stem cell population was notdepleted, arguing against these cells being the stem cells (Morsehead etal., Neuron 13:1071).

[0008] WO 97/44442 (Johe) discloses isolation of stem cells from the CNSof mammals and more specifically from the subependymal region ofstriatum lining the lateral ventricles. However, only subependymal cellsare used and thus there is no further teaching regarding the identityand role of mammalian ependymal cells that alters the conventional one.

[0009] WO 95/13364 (Weiss, et al.) relates to a method of proliferationof CNS precursor, cells located by the CNS ventricle of a mammal.However, only precursor cells are disclosed, and there are no teachingsregarding other cell stages, such as stem cells.

[0010] In this context, it is interesting to note that besides theolfactory bulb and the hippocampus, data on continuous neurogenesisthroughout adulthood in other regions of the mammalian brain have beenscarce. As an example that neurogenesis may be a more widespreadphenomenon, a small number of cells with the capacity to generateneurons in vitro has been isolated from the striatum and septum (Palmeret al., Mol. Cell. Neurosci. 6:474), although it has not been tested ifthese cells have stem cell properties or if they are committed neuronalprogenitors.

[0011] There is increasing evidence that nervous system injuries mayaffect stem cells in the adult CNS. After both spinal cord and braininjuries, nestin expression is increased in cells lining the centralcanal and in the subventricular zone, respectively (Frisén et al., J.Cell Biol. 131:453, Holmin et al. Eur. J. Neurosci. 9:65). These cellshave been suggested to derive from stem cells. With time, nestinexpressing cells are seen progressively further from the central canaland the lateral ventricle and these cells express astrocytic markers(Frisén et al., J. Cell Biol. 131:453, Holmin et al. Eur. J. Neurosci.9:65). These data have lead to the suggestion that stem cells orprogenitor cells residing by the ventricular system are induced toproliferate, migrate toward the site of the injury and differentiate toastrocytes. Furthermore, hippocampal lesions increase the proliferationof hippocampal progenitor cells and the number of granular neurons inthe hippocampus (Gould et al. Neurosci. 80:427). However, since the stemcell has not been identified or exactly localized it is not clearwhether stem cells play a role in injury processes.

[0012] Cell loss is a common factor in many types of nervous systemdisorders. Distinct cell types are affected in different diseases, e.g.dopaminergic neurons in Parkinson's disease, motor neurons inamyotrophic lateral sclerosis and oligodendrocytes in multiplesclerosis. Several different cell types in a certain area can beaffected in other situations, such as stroke or traumatic injury.Currently, no methods are available in clinical practice to stimulategeneration of new cells in the nervous system. Transplantation of cellsfrom human embryos or animals have been tested clinically with someencouraging results. However, these methods have several problems,mainly ethical and immunological, which makes it very unlikely that theywill be used in any larger number of patients.

[0013] Accordingly, the discovery of the existence of neural stem cellsin the adult CNS of mammals is important and may make it possible todevelop strategies to stimulate generation of new neurons or glialcells. However, several important questions have remained unanswered andbetter methods to culture these cells and to study them quantitativelyin vivo are needed. Most importantly, it is absolutely vital to identifyand localize the stem cell in the adult CNS in order to be able to studythese cells further and to stimulate generation of new neurons from thestem cells.

[0014] Furthermore, there are no methods available today to purify stemcells at an early step in tissue culture. Although there are severalgeneral methods available for purifying cell populations in othertissues, it is impossible to utilize these methods, or to develop newmethods, without knowledge of the true identity of the stem cell. Suchmethods would allow studies of a more well defined cell population andwould be valuable for screening pharmaceutical compounds. Moreover, thedevelopment of quantitative methods to label and follow the stem cellsand their progeny in vivo to allow detailed studies of, for example,regulation of the generation of new neurons to analyze the effect ofdifferent chemicals or genetically manipulate the stem cells are needed.Again, although there are methods known in the art that can be used tofollow other cell populations in vivo, it is impossible to utilize thesemethods or develop new methods for following stem cells since theidentity of the stem cell has been unknown. The development ofquantitative methods to follow stem cells and their progeny in animalmodels of neurodegenerative disorders and injuries of the CNS would openup the possibility to screen new treatment strategies in humanconditions where today only some of the symptoms, but not the neuronalloss per se, can be alleviated.

[0015] Thus, a problem within this field is that even though neural stemcells are known to exist, the localization and identity therof is notknown. Could this be accomplished, a great step forward would be takenby research aimed at providing the above defined goals.

SUMMARY OF THE INVENTION

[0016] The object of the present invention is solve the above definedproblem. More specifically, the present invention relates to a method ofisolating ependymal neural stem cells from a mammalian CNS. The presentidentification of the stem cell has now made it possible to developmethods to purify these cells, study them quantitatively in vivo,genetically modify them and stimulate them with various pharmaceuticalcompounds in vitro and in vivo. Furthermore, the present inventionprovides evidence that the stem cells follow new migratory pathways tovarious neuroanatomical cell groups in the CNS and that they are able totransform into neurons in vivo. The invention also comprises an unbiasedquantitative method to assess neurogenesis in various regions of thebrain as well as techniques to analyze the total number of stem cellsand their progeny migrating to various regions of the brain. Altogether,the developments provided by the present invention greatly increase thepossibilities to develop strategies to stimulate generation of new cellsin the central nervous system.

[0017] Accordingly, the actual identity and localization of ependymalneural stem cells are disclosed herein for the first time ever, thusenabling various advantageous uses and applications thereof within themedical and diagnostic field. The invention also relates to in vitro andin vivo assays, wherein the new findings according to the invention areadvantageously employed.

BRIEF DESCRIPTION OF THE DRAWINGS

[0018]FIG. 1 illustrates the specific labeling of ependymal cells and isa schematic drawing of the migration of neurons in the adult forebrainand the structure of the wall of the lateral ventricle.

[0019]FIG. 2 illustrates the generation of olfactory bulb neurons andneurospheres.

[0020]FIG. 3 illustrates the enrichment of neural stem cells with anependymal cell specific marker.

[0021]FIG. 4 illustrates the proliferation of ependymal cells by showingan immunohistochemical detection of 5-bromo-2′-deoxyuridine (BrdU) inthe lateral wall of the lateral ventricle after two weeks continuousBrdU administration (A,B) or two weeks administration followed by oneweek without BrdU (C,D).

[0022]FIG. 5 discloses how the ependymal cell proliferation is inducedby injury.

[0023]FIG. 6 shows the generation of astrocytes from ependymal cellsafter spinal cord injury.

[0024]FIG. 7 discloses transplantation of purified of stem cells derivedfrom transgenic animals.

[0025]FIG. 8 shows the generation of neurons in substantia nigra fromstem cells in the adult mouse.

[0026]FIG. 9 illustrates migratory streams of stem cells or theirprogeny in the midbrain.

[0027]FIG. 10 discloses the generation of neurons in hippocampus fromstem cells localized in the ependyma.

DEFINITIONS

[0028] As used herein, the term “isolated” refers to cell fractionsisolated from an animal, (e.g., a human, a rat, a mouse, etc.) andpurified up to at least about 10%, such as 80%. Purity is measured bycomparing the number of neural stem cells with the total number ofcells. For example, an “80% pure” preparation of ependymal neural stemcells means that 80% of the cells in the preparation are ependymalneural stem cells. The term “neural stem cells” relates to cells capableof generating aggregates of undifferentiated cells, so calledneurospheres, under suitable conditions, e.g. a medium containingappropriate mitogens. “Ependymal cells” refers to any cell originatingfrom the ependymal layer in the CNS ventricular system or the same celltype located elsewhere. In the present context, it is to be understoodthat among the features that characterize the ependymal neural stemcells according to the invention is the capability thereof to generatenew stem cells, precursors, progenitor cells, neurons, astroglia oroligodendroglia. The term “adult” is used herein to differentiate theneural stem cells previously identified in embryos from the presentependymal neural stem cells of the invention obtained from post-natalmammals. Thus, adult stem cells are in essence non-embryonic stem cells.

DETAILED DESCRIPTION OF THE INVENTION

[0029] More specifically, in a first aspect, the present inventionrelates to a method of isolating ependymal neural CNS stem cells from ananimal, which method comprises the steps of

[0030] (a) screening single cells obtained by dissociating CNS tissuefrom said animal for cells exhibiting at least one characteristic of anependymal neural stem cell; and

[0031] (b) recovering the cells that exhibit the characteristic orcharacteristics screened for in step (a).

[0032] The animal from which the cells are isolated may be a human. Inan advantageous embodiment of the present method, the cells screened instep (a) are from tissue comprising the walls of the ventricular systemof the brain or spinal cord, or any other area that contains ependymalcells, of said animal. The dissection and recovery of such tissue iseasily performed by the skilled man in this field by any suitableroutine method. The dissociation of the tissue into individual cells isperformed by any suitable method, such as an enzymatic and/or mechanicaltreatment, and is not restricted in any way as long as the desiredsingle cells are obtained as a result thereof. Examples of such methodsare e.g. trituration, trypsin treatment, collagenase treatment andhyaluronidase treatment. Most preferably, the dissociation is performedby enzymatic treatment with trypsin. The dissociation of tissue mayalternatively be performed by any other method easily chosen by theskilled man in view of the prevailing conditions.

[0033] The screening of the resulting cells is also performed by anysuitable method depending on the characteristic, trait or property of anependymal cell used. In one embodiment of the present method, thescreening is performed by use of the expression of a specific cellsurface marker, such as a protein. Such an expression of a surfaceprotein may for example be the expression of the Notch1, Notch2 and/orNotch3 receptors. In the most preferred embodiment of this method, thesingle cells are screened for their expression of the Notch1 receptor.In an alternative embodiment of this aspect of the invention, the singlecells are screened for by specifically labeling ependymal neural stemcells or ependymal cells and choosing so labeled cells. Such a labelingmay be a dye and is advantageously a fluorescent labeling, such as DiI,as shown in example 1. However, in an alternative embodiment a virus,such as an adenovirus, may be used to label the cells. The labeling ofcells is used extensively within research and diagnostic methods and thechoice of a suitable technique is thus easily within the skill of one inthe art.

[0034] In a preferred embodiment-of the method according to theinvention, the cells recovered from step (b) are comprised of at leastabout 10% of ependymal neural stem cells, such as 10-50%, e.g., about35%, or in a preferred embodiment, up to about 90%, or most preferablyan essentially pure culture of ependymal neural stem cells. Naturally,high concentrations are possible to obtain, depending on the screeningmethod chosen. Previously, in prior art procedures, parts of a brainhave been dissociated and specific growth factors have been added inorder to induce growth of a specific cell type. Such procedures havenever been aimed at obtaining a pure population of ependymal neural stemcells at an early step in the culture procedure, since the identity andcharacteristics (for example expression of specific cell surfacemarkers) of the ependymal neural stem cell have been unknown before thepresent invention. Thus, in practice, the present method yields thedesired concentration of a cell type, i.e. the ependymal neural stemcells disclosed herein, that has never been identified and/or localizedbefore. In a specific embodiment, the product consists of about 90-95%of ependymal neural stem cells. In one advantageous embodiment, theproduct of the method is a cell fraction consisting almost entirely,that is, of about 100%, of the ependymal neural stem cells. Accordingly,the present invention also relates to isolated ependymal neural stemcells obtainable by the method according to the present invention aswell as to any fraction of isolated ependymal neural stem cells.

[0035] Thus, the invention relates to an ependymal neural stem cell ofpost-natal or adult tissue from the CNS or the ventricular system of thebrain or spinal cord. Preferably, the ependymal neural stem cellaccording to the present invention expresses a cell surface marker, suchas a protein, and most preferably it expresses Notch1.

[0036] In a second aspect, the present invention relates to geneticallymodified ependymal neural stem cells. Manipulations may be performed inorder to modify various properties of the cell, e.g. to render it moreadapted or resistant to certain environmental conditions, to induce aproduction of one or more certain substances therefrom, which substancesmay e.g. improve the viability of the cell or alternatively may beuseful as drugs or medicaments. The invention of methods to purifyependymal neural stem cells in cell culture allows for all types ofgenetic manipulation, for example transfection of these cells withplasmid or viral expression vectors or purification of cells fromtransgenic organisms or suppression of gene expression with for exampleantisense DNA or RNA fragments. Localization of the ependymal neuralstem cell in vivo allows for alteration of gene expression in thesecells in situ with for example viral vectors.

[0037] Altering the expression of genes in cells can make these cellsproduce a given protein of choice or can prevent the production of anunwanted protein. Manipulating the genes of cells in vitro or in vivo inaccordance with the present invention may be beneficial in a widevariety of situations. For example, cells engineered to express a growthfactor, cytokine, hormone or any other protein can be transplanted toindividuals which may need continuous administration of such a proteinto stimulate e.g. cell signaling or cell survival. The cells will thusserve as continuous administrators of a pharmaceutical substance. Cellsfor such use can be genetically tailored, by e.g. transfection withplasmid or viral vectors, or the cells can be taken from transgenicorganisms. Transgenic organisms comprising cells according to thepresent invention are also within the scope of the present invention.Furthermore, gene expression can be altered in situ in an organism byinducing ectopic gene expression with plasmid or viral vectors as wellas antisense DNA or RNA fragments. Under certain conditions, it may bevaluable to use cells which lack a certain gene or produces lower levelsof the gene product. For example, transplantation of cells or tissuesbetween different individuals is limited by the expression of certainproteins on the surfaces of cells which induces the host immune systemreject the graft. This is a major problem, especially if the twoindividuals are of different species. One way to circumvent this problemis to generate genetically modified cells or animals that lack genesthat induce rejection by a host immune system. Other importantimplications for manipulating gene expression in cells in vitro or invivo include inducing differentiation of an undifferentiated cell towarda certain cell fate or stimulating survival of the cell by suppressingintrinsic or extrinsic cell death signals. Furthermore, by introducingcertain genes it is possible to immortalize cells and generate clonalcell lines with special features. Since the identity and localization ofneural stem cells in the adult central nervous system has been unknown,it has previously been difficult to modify these cells genetically,especially in vivo (for reviews of gene therapy procedures, seeAnderson, science 256:808; Nabel and Felgner TIBTECH 11:211; Mitani andCaskey TIBTECH 11: 162; Mulligan Science 926; Dillon TIBTECH 11: 167;Miller Nature 357:455; Van Brunt Biotechnology 6(10):1149; VigneRestorative Neurology and Neuroscience 8:35; Kremer and PerricaudetBritish Medical Bulletin 51(1) 31; Haddada et al. in Current Topics inMicrobiology and Immunology, Doerfler and Böhm (eds) Springer-Verlag,Heidelberg Germany; and Yu et al., Gene Therapy 1:13, each of which isincorporated herein by reference). Thus, the present invention alsoencompasses gene therapy methods, wherein ependymal neural stem cellsare used as well as preparations intended to be used in such methodscomprising the cells according to the invention. Such gene therapymethods may be used to treat and/or prevent any conditions wherein theneurons or glia in the CNS have been impaired or are defective.

[0038] In another aspect, -the present invention relates to an ependymalneural stem cell for use in therapy, e.g. as a medicament. In addition,the invention also relates to the use of an ependymal neural stem cellin the preparation of a medicament for regulating the neurogenesis orgliogenesis in the central nervous system, such as the brain. Suchregulation is either inducing or inhibiting and the treatment may beaimed at Parkinson's disease, Alzheimer's disease, stroke, trauma etc.In the case of glial cells, the medicament may be intended for treatingmultiple sclerosis and other glia related conditions. In one particularembodiment of the invention, these aspects of the invention useependymal neural stem cells obtained by the method disclosed above, eventhough the invention also encompasses the uses of any ependymal neuralstem cells, such as genetically modified ependymal neural stem cells,for the present purposes.

[0039] In a further aspect, the invention relates to a pharmaceuticalpreparation comprising at least one ependynmal neural stem cellaccording to the invention and a pharmaceutically acceptable carrier.The preparations according to the invention may be adapted for injectioninto a suitable part of the central nervous system. Such apharmaceutical preparation comprises any suitable carrier, such as anaqueous carrier, e.g. buffered saline etc. The active composition of thepresent preparation is generally sterile and free of any undesirablematter. In addition, the preparations may contain pharmaceuticallyacceptable auxiliary substances as required to approximate physiologicalconditions, such as pH adjusting agents etc. The concentration of thepresent ependymal neural stem cell in the preparation will varydepending on the intended application thereof and the dosages thereofare decided accordingly by the patient's physician. The pharmaceuticalcompositions of the present invention comprise about 10³ to 10⁹ependymal neural stem cells. In some preferred embodiments, thecompositions comprise about 10⁵ to 10⁸ ependymal neural stem cells. Insome preferred embodiments, the compositions comprise about 10⁷ependymal neural stem cells. The stem cells used may have been isolatedby the present method or any other suitable method or obtained in anyother way. In a preferred embodiment, the present ependymal neural stemcell may have been genetically manipulated in order to be especiallyadapted for the intended use thereof. In a further aspect, the presentinvention also relates to an animal, such as a mouse, that comprises agenetically modified ependymal neural stem cell according to theinvention. Such animals may, e.g., be useful as models in research orfor the testing of drugs.

[0040] In yet a further aspect, the present invention relates to methodsof using the present ependymal neural stem cells as “drug targets”,preferably in in vitro assays, to stimulate or inhibit ependymal neuralstem cell proliferation or differentiation into particular neuronalphenotype or glial subtype. In a particular embodiment, the inventionrelates to a method of screening for differentiation inducing agents,the method comprising culturing ependymal neural stem cells in vitro,exposing the cells to one or more potential differentiating agents, andassaying for one or more indicators of differentiation. Screening forinhibitors of differentiation can be performed by, for example,culturing isolated ependymal neural stem cells, exposing the cells toone or more known differentiating agents, and either before,concomitantly with, or after exposure to the differentiating agent oragents, exposing the cells to one or more potential inhibitors ofdifferentiation and assaying for one or more indicators ofdifferentiation.

[0041] When screening for potential proliferation inducing agents, themethod may, e.g., involve culturing of isolated ependymal neural stemcells, exposing the cells to one or more potential proliferationinducing agents, and assaying for enhanced neural stem cell growth.Screening for inhibitors of proliferation can be performed by, forexample, culturing isolated ependymal neural stem cells, optionallyexposing the cells to one or more known proliferation inducing agents,and either before, concomitantly with, or after exposure to theproliferation inducing agent or agents, if used, exposing the cells toone or more potential inhibitors of proliferation and assaying for oneor more indicators of proliferation. The present invention also relatesto the substances obtained by the methods defined above.

[0042] In another aspect, the present invention relates to an unbiasedquantitative or qualitative, preferably quantitative, method to assessneurogenesis and migratory streams of stem cell progeny, preferably inin vivo assays, in various regions of the brain as well as techniques toanalyze the total number of stem cells and their progeny migrating tovarious regions of the brain. This is for the development of newscreening methods, which methods are also within the scope of thepresent invention as defined by the appended claims. This could be ofuse in diagnosing patients suffering from neurodegenerative diseases, ifthe development of ependymal cell markers suitable for positron emissiontomography (PET), or other imaging systems able to visualize the livingbrain with sufficient resolution, allows the diagnosis of defectivemigration and/or differentiation of the stem cell progeny in human CNS.The present invention also encompasses the diagnostic use of labeledstem cells in the ependymal layer in humans. Such cells can be followedwith an imaging system (e.g., by PET) to assess their migratory pattern.The cells may be labeled by, for example, DiI ventricular injection.Cells may be labeled in vitro, then injected, or labeled in vivo, as isdescribed in the examples. Thus, the present invention also relates tokits and assays for performing such methods as well as to substancesobtained by the present methods.

[0043] In a last aspect, the present invention relates to a method oftreating a patient suffering from a neurodegenerative disease, whichmethod comprises the administration to said patient of apharmaceutically effective amount of ependymal neural stem cellsaccording to the invention. The patient may be any animal, including ahuman. There are several potential injection sites. Thus, the cellscould be injected into the nerve terminal area of the cells thatdegenerate in the particular neurodegenerative disorder. For example, inParkinson's Disease, the dopamine neurons that die are situated in themidbrain in substantia nigra pars compacta, but the cells can betransplanted into the nerve terminal area in the forebrain.Alternatively, they may be transplanted directly into the ventricularsystem, into the migratory streams of cells described in the examplesbelow, or in the neuronal cell body region of the, cells that degeneratein the particular human neurodegnerative disorder. In general, such amethod is based on administration of stem cells with an unimpairedfunction and ability to produce neurons or other cell types depending onthe human CNS disorder. Alternatively, neurons or glial cells generatedfrom stem cells in vitro can be administrated to the CNS. Methods fortransplanting cells into the brain have been described, and are known toone of skill in the art (Widner, et al., New England J. Med., 327:1556;Wenning, et al., 1997, Ann. Neurol., 42(1):95-107; Lindvall, et al.,1994, Ann. Neurol., 35(2):172-80; Widner, et al., 1993, Acta NeurolScand Suppl, 146:43-5; Neural Grafting in the Mammalian CNS, 1985,Bjorklund and Stenevi, eds; U.S. Pat. No. 5,650,148; InternationalPatent Publication WO 9206702, Itukura, T., et al., 1988, J. Neurosurg.68:955-959, each of which are incorporated herein by reference).

[0044] In an alternative embodiment, the invention relates to a methodof treatment and/or prevention of neurodegenerative disorders in a humanor animal patient, wherein the existing defective neural stem cells'ability to produce new neurons or migrate to the appropriate target isrestored. Such a method is based on the administration of a substancethat stimulates and induces the neural stem cells' native properties andcapability to produce neurons. Alternatively, such a method may be basedon the administration of a substance that actually inhibits thedegenerative process of the neurons.

[0045] In summary, the present invention will make it possible todevelop new treatment strategies in diverse diseases of the CNS, notonly in diseases with a slow progression of the neurodegeneration(including Alzheimer's disease, Parkinson's disease, amyotrophic lateralsclerosis, multiple sclerosis) but also in clinical situations of acutetrauma to the head or spinal cord as well as in cerebrovasculardiseases. Our finding that stem cells may transform into severaldifferent neuronal phenotypes (dopamine neurons, GABA-neurons, serotoninneurons), open up possible applications beyond the above mentioneddiseases, where cell loss is central to the development of the disease,into possible new areas, including depression and other mentaldisorders.

DETAILED DESCRIPTION OF THE DRAWINGS

[0046]FIG. 1: Specific Labeling of Ependymal Cells.

[0047] Schematic drawing of the migration of neurons in the adultforebrain and the structure of the wall of the lateral ventricle (A).The ventricle (V) is lined by ependymal cells (E). Between the ependymallayer and the striatum (S) is the subventricular zone (SVZ), whereprecursor cells (light blue) divide to give rise to immature neurons(dark blue). The neurons migrate to the olfactory bulb (blue arrow). (B)Labeling of ependymal cells. DiI is injected stereotaxically into alateral ventricle, resulting in labeling of ependymal cell throughoutthe ventricular system. In some of these animals, an incision (gray areain the spinal cord cross section) was made in spinal cord dorsalfuniculus. The DiI injection labels the ependymal layer lining thelateral ventricle (C, D) and the spinal cord central canal (E) six hoursafter the injection. The choroid plexus (CP) is labeled in (C).

[0048]FIG. 2: Generation of Olfactory Bulb Neurons and Neurospheres

[0049] Ten days after injection of DiI (A, C) or replication-deficientadenovirus expressing LacZ (B, D) into the contralateral lateralventricle, labeled cells are seen in the subventricular zone (A, B) andolfactory bulb (C, D). The inset in (C) shows DiI in aβIII-tubulin-immunoreactive neuron. Bright-field (E) and fluorescence(F) micrographs showing neurospheres from the brain of an animal whichhad received an intraventricular DiI injection. Two very weakly labeledor unlabeled neurospheres are indicated with arrowheads.

[0050]FIG. 3: Enrichment of Ependymal Neural Stem Cells with anEpendymal Cell Specific Marker

[0051] Immunofluorescence localization of Notch1 in the wall of thelateral ventricle (A) and in the spinal cord (B). Notch1immunoreactivity is restricted to ependymal cells lining the lateralventricle and central canal. The selective localization of Notch1 toependymal cells enabled enrichment of ependymal cells from acutelydissociated brain and spinal cord tissue. The dissociated cells wereincubated with antiserum raised against Notch1, followed by incubationwith magnetic bead conjugated secondary antibodies and magneticseparation of labeled (Notch1 fraction) and unlabeled cells (washfraction). In control experiments, the primary antiserum was omitted.The number of cells in each fraction was calculated and the number ofneurospheres generated in the different cultures was counted (C).

[0052]FIG. 4: Proliferation of Ependymal Cells

[0053] Immunohistochemical detection of BrdU in the lateral wall of thelateral ventricle after two weeks continuous BrdU administration (A, B)or two weeks administration followed by one week without BrdU (C, D).(B) and (D) show details from (A) and (C), respectively. Labeledependymal cells are indicated with arrowheads in (B) and (D).

[0054]FIG. 5: Ependymal Cell Proliferation is Induced by Injury

[0055] Immunohistochemical detection of BrdU in the spinal cord after 8hours (A, D-F) or two weeks administration (B, C) of BrdU. (G)Proportion of spinal cord ependymal cells incorporating BrdUadministered during the last 8 hours before sacrifice (BrdU labelednuclei/total number of ependymal cell nuclei visualized with propidiumiodide, n=3-5 rats at each time point, error bars show SEM).

[0056]FIG. 6: Generation of Astrocytes from Ependymal Cells after SpinalCord Injury

[0057] Distribution of DiI, nestin- and GFAP-immunoreactivity in thespinal cord. The animal in (D-F) was subjected to a dorsal funiculusincision 4 weeks prior to analysis. All animals received anintraventricular DiI injection prior to injury. DiI andnestin-immunoreactivity is shown in the same sections, andGFAP-immunoreactivity in an adjacent section in (A-F). The approximatedelineation of the injured area is indicated by the broken line in (D).(G) shows DiI (red) and GFAP-immunoreactivity (green) in the dorsalfuniculus 2 weeks after the lesion. A yellow signal indicatesco-localization of DiI and GFAP-immunoreactivity. (H) Confocal laserscanning microscope visualization of DiI and GFAP-immunoreactivity inthe scar tissue 2 weeks after injury. Two GFAP-immunoreactive DiIlabeled cells are indicated by arrowheads, and a DiI labeled cell whichdoes not show any detectable GFAP is indicated with an arrow.

[0058]FIG. 7: Transplantation of Ependymal Neural Stem Cells

[0059] Transplantation of purified stem cells derived from transgenicmice expressing LacZ to the striatum of adult rats. The arrows point toa group of grafted cells. (B) shows a detail from (A).

[0060]FIG. 8: Generation of Neurons in Substantia Nigra from Stem Cellsin the Ependymal Layer in the Adult Rat.

[0061] Microphotograph of nigral tyrosine hydroxylase-positive neurons(green) in substantia nigra pars compacta also labeled with DiI (red) inrodents after administration of this fluorescent dye to the adult animalfour months earlier. Arrows point at two nigral dopamine neuronscontaining the fluorescent marker labeling ependymal, neural stem cells.

[0062]FIG. 9: Migratory Streams of Stem Cells from the Ependymal Layeror Their Progeny in the Mouse Midbrain.

[0063] Arrows point at the ventromedial migratory streams (red cells) ofthe DiI labeled ependymal cells that reach the medial substantia nigrapars compacta (*). SNR=substantia nigra pars reticulata,IP=Interpeduncular nucleus. Arrows show lateral (1) and ventral (v)directions. Several pathways reaching rostral, caudal, medial andlateral parts of substantia nigra pars compacta respectively wereidentified. In addition to the illustrated ventromedial stream, adorsolateral and a midline stream were identified.

[0064]FIG. 10: Generation of Neurons in Mouse Hippocampus from StemCells Localized in the Ependymal Layer.

[0065] Microphotograph illustrating that the DiI labeled ependymal cellsor their progeny migrate to the granule cell layer of the dentate gyrus(DG) of hippocampus. Arrows show lateral (1) and ventral (v) directions.

[0066] The following examples are presented only as illustrating theinvention as defined by the claims and are in no way intended to limitthe scope thereof. All references made below and elsewhere in thepresent disclosure are hereby included herein by reference.

EXAMPLES

[0067] Labeling of Ependymal Cells and Their Progeny in vivo

[0068] To test whether neurons may be generated from ependymal cells, weinjected the fluorescent label DiI or a replication deficient adenovirusexpressing the reporter-gene LacZ into the lateral ventricles of adultrats or mice. Male Sprague-Dawley rats weighing 280-320 g or maleC57BL/6 mice weighing 25-30 g were anaesthetized with chloral hydrate(400 mg/kg). Unilateral stereotaxic injections of 10 μl (rats) or 3 μl(mice) of 0.2% w/v DiI (Molecular Probes) in DMSO or 50 μl adenovirussolution (containing 10⁹ plaque forming units) were made 0.9 mm (rats)or 0.5 mm (mice) posterior and 1.4 mm (rats) or 0.7 mm (mice) lateral toBregma and 4 mm (rats) or 2 mm (mice) below the dura mater into thelateral ventricle. The injections resulted in specific labeling of theependymal layer throughout the ventricular system; no labeling was seenin the subventricular zone nor in the brain parenchyma (FIG. 1). Thus,this method makes it possible to specifically follow the fate of thelabeled ependymal cells and their progeny. Analysis of the distributionof DiI revealed an increasing number of labeled cells in the rostralmigratory stream (Goldman et al., Trends Neurosci. 21:108), and after 10days the first DiI labeled neurons were seen in the olfactory bulb, aregion where new neurons are added continuously in adult mammals (FIG.2). Similarly, in animals injected with the adenovirus, LacZ expressingcells were found in the olfactory bulb from 10 days after the injection,albeit at much lower numbers than in the DiI injected animals asexpected since the adenovirus is replication deficient and the labelwill thus only be inherited by a subset of the progeny of the infectedcell (FIG. 2). LacZ expression was detected with X-gal staining asdescribed (Park et al., EMBO J. 16:3106).

[0069] Identification and Localization of Ependymal Neural Stem Cells byCulturing Labeled Ependymal Cells

[0070] The specific labeling of ependyma in vivo may be used to testwhether the labeled cells have stem cell properties in vitro. After aninjection of DiI (described above), rats were killed with CO₂, theirbrains removed and kept in ice-cold PBS. The lateral walls of thelateral ventricles were dissected out. The tissue was minced withscissors, shifted to 4 ml of dissociation medium (0.075% collagenasetype 1 (Worthington), 0.075% hyaluronidase (Sigma), 2000U DNAse I in 4ml 0.2M PIPES (Sigma) and incubated at 37° C. for 30 min. Mechanicaldissociation by gentle successive trituration through a 5 ml and 1 mlPasteur pipette was followed by leaving the suspension for 2 min,allowing larger fragments to settle to the bottom. Then the supernatantwas passed through a 40 μm mesh (Falcon). To the filtered suspension, 4ml of cold medium (DMEM/F12) was added. Cells were spun down at 200 gfor 4 min. The supernatant was removed, the pellet resuspended in 10 mlsucrose solution (0.9M in 0.5×HBSS) and centrifuged for 10 min at 750 g.The supernatant was removed and the pellet resuspended in 2 ml ofculture medium, placed on top of 10 ml 4% BSA in EBSS solution andcentrifuged at 200 g for 7 min, followed by a washing step in DMEM/F12.The culture medium was made of: 0.5 ml L-glutamine, 0.75 ml 1M HEPES (15mM), 50 μl 20 μg/ml EGF (20 ng/ml), 1 ml B27 supplement, 0.5 ml100×penicillin/streptomycin stock and, finally, DMEM-F12 medium to atotal volume of 50 ml. Cell cultures were maintained at 37° C., 5% CO₂in a humid atmosphere.

[0071] Under these conditions, characteristic spheroid cell aggregatesof undifferentiated cells were formed in the cultures. These cellaggregates derive from a single stem cell and thus represent a clone ofcells. Of these spheres, 88.6±1.20% and 89.0±1.23% from lateralventricle and spinal cord, respectively, were clearly DiI labeled (meanfrom 5 independent experiments±SEM). DiI labeled spheres were collectedand dissociated to single cells. Many of these cells formed new spheres,and when induced to differentiate by adding serum to the medium, most ofthese secondary spheres generated neurons, astrocytes andoligodendrocytes. Generation of differentiated progeny was demonstratedby immunohistochemical labeling with the following cell type specificantibodies: anti-glial fibrillary acidic protein (Dako) for astrocytes,Tuj1 (Babco) for neurons and O4 (Boehringer Mannheim) foroligodendrocytes. These experiments identify a useful method to studyependymal cells in vitro and demonstrate that ependymal cells have selfrenewal capacity and that they are multipotent, i.e. they are bona fidestem cells.

[0072] Purification of Ependymal Neural Stem Cells by Cell Sorting

[0073] We have found that Notch1 protein, a cell surface receptorexpressed in the nervous system during embryonic development (Kopan etal., Trends Genet. 13:465), is selectively expressed in ependymal cellsbut not in subventricular zone cells in the adult rat brain and spinalcord (FIG. 3). We took advantage of this selective expression of Notch1to isolate ependymal cells from acutely dissociated lateral ventriclewall and spinal cord tissue by magnetic sorting with Notch1 antiserum.For magnetic sorting, cells were collected as above and resuspended in100 μl culture medium of the above defined composition, 1 μl of rabbitantiserum raised against Notch1 was added and incubated at 4° C. for 20min. Subsequently, cells were washed with 6 ml of DMEM/F12, the pelletwas resuspended in 100 μl of culture medium and 30 μl of pre-washed(with 0.5% BSA in PBS) magnetic bead-conjugated anti-rabbit antiserum(1.8-2.1×10⁷ beads, Dynal) was added and incubated for 20 min at 4° C.with occasional shaking. After incubation, 2 ml of culture medium wasadded, the suspension transferred into a 2 ml Eppendorf tube, placed ina Dynal magnetic separator and left for 2 min. The supernatant wascollected in a 35 mm uncoated Nunc dish (‘wash’ fraction), then themagnet was removed from the separator, 2 ml culture medium (as definedabove) was added to resuspend the bead-cell suspension and the magneticseparation step was repeated. The supernatant was again transferred to aculture plate. After removal of the magnet, 2 ml of culture medium wereadded, the remaining cells resuspended and transferred to a 35 mmuncoated Nunc dish. Throughout the whole procedure, all solutions andthe cell suspensions were kept cold. Cell cultures were maintained at37° C., 5% CO₂ in a humid atmosphere. Culture medium (composition asdescribed above) was renewed every 3-4 days. Cells which had magneticbeads attached (sorted) or not (wash fraction) were then cultured andassayed for the presence of stem cells. Neurospheres started to appeararound day 4-5 after isolation. In experiments where the cells had beensorted with the Notch1 antiserum, but not in experiments where theNotch1 antiserum was omitted, the majority of spheres formed in thesorted fraction (FIG. 3). When spheres formed in the Notch1 sortedfraction were dissociated they formed secondary spheres which weremultipotent corroborating that ependymal cells are neural stem cells.

[0074] In other experiments, in vivo labeling of ependymal cells (seeabove) with DiI was followed by fluorescence activated cell sorting(FACS) or manual picking of fluorescent cells and resulted in highlyenriched cultures of ependymal cells.

[0075] Ependymal Cells have a Slow Proliferation Rate and Generate aTransit Amplifying Precursor Population

[0076] Previous studies, based on the lack of incorporation of labelednucleotides after a single or a few injections, have indicated thatependymal cells do not proliferate in adult mammals. A characteristicfeature of stem cells is that they proliferate slowly or rarely andadministration of labeled nucleotides over long time periods have beenused to identify slowly cycling stem cells in other tissues. It is thuslikely that a slow proliferation rate of ependymal cells, which onewould expect if they are stem cells, would be missed if analyzed by asingle or a few injections of labeled nucleotides.

[0077] In order to characterize the proliferation of ependymal cells, wesupplied the thymidine analogue 5-bromo-2′-deoxyuridine (BrdU, Sigma)continuously over long time periods. Rather than doing repeatedinjections, we administered BrdU to adult mice through; the drinkingwater over a two to six week period before analysis. To achieve longterm labeling of the mouse brain, we added 1 mg/ml BrdU to the drinkingwater of mice. The water was exchanged twice a week and protected fromlight with aluminum foil. BrdU was efficiently taken up through theintestine, and resulted in labeling of ependymal cells lining thelateral walls of the lateral ventricles. Ependymal cells lining the roofand the medial wall of the lateral ventricles were rarely labeled,corresponding to the lateral wall being the most active neurogenicregion. Very large numbers of BrdU labeled cells were present in thesubventricular zone (FIG. 4). The labeled cells in the subventricularzone were often grouped in tight cell clusters, giving the impression ofbeing a clone of cells (FIG. 4). Strikingly, in many cases such a cellcluster was located in close proximity to a labeled ependymal cell (FIG.4). The proliferating precursor cells migrate closely together in thesubventricular zone to the antero-lateral tip of the lateral ventriclewhere they enter the rostral migratory stream. The spatial relationshipbetween a labeled ependymal cell and a cluster of subventricular zonecells was in the vast majority of cases so that the subependymal cellswere shifted toward the rostral migratory stream in relation to thelabeled ependymal cell (FIG. 4).

[0078] The fact that stem cells have a low proliferation rate has beenused to localize stem cells in other tissues. When labeled nucleotidesare administered over prolonged time periods, both rapidly and slowlyproliferating cells will be labeled. By letting the animals survive fora period after the administration of the labeled nucleotide, rapidlyproliferating cells will be given time to dilute the label by continueddivisions or by migrating away. Therefore, only slowly proliferatingcells will retain the label with time. We analyzed animals which hadreceived BrdU continuously over a two to six week period followed by 2weeks without BrdU. In these animals, very few labeled cells were seenin the subventricular zone, indicating that the vast majority of cellshad diluted the label by repeated divisions or migrated away (FIG. 4).However, a substantial number of ependymal cells were still labeled(FIG. 4).

[0079] We next studied the proliferation of spinal cord ependymal cells.A substantial number of ependymal cells lining the central canal werelabeled after prolonged administration of BrdU through the drinkingwater (FIG. 5). In contrast to in the lateral ventricle subventricularzone, few labeled cells were seen just outside the central canalependyma (FIG. 5). However,the few labeled cells that were seen close tothe central canal often resided in close proximity with a labeledependymal cell, suggesting that this cell may derive from the ependyma(FIG. 5).

[0080] A Quantitative Method Analyzing Neurogenesis in vivo in a DefinedBrain Region Such as Substantia Nigra Pars Compacta in the Midbrain

[0081] Several unpublished data from the present inventors have promptedthem to postulate that the region of the brain where dopamine neuronsdie in Parkinson's disease, i.e. substantia nigra (Date, Brain Res.Bull. 40:1) represents a new region where a continuous turnover ofneurons is present. State of the art stereological cell countingtechniques in situ have been utilized and further developed (Gundersenet al., APMIS 96:857; Janson et al., Neuroscience 57:931) and the totalnumber of nigral neurons in young and aged mice, as well as the totalnumber of apoptotic neurons in the same region, analyzed. Briefly, thepresent inventor's unpublished results (Janson et al.) indicate thatyoung and aged mice have the same number of nigral dopamine neurons,although a low number of neurons die spontaneously through apoptosis. Atthe same time the present inventors have found that nestin, a marker forneuronal progenitor cells as described above, is present in asubpopulation of nigral neurons (unpublished data, Janson et al.). Takentogether these data indicate the possibility of a continuousneurogenesis in balance with neuronal apoptosis, i.e. neuronal turnover,which is described below. The quantitative method allows in vivoscreening of substances enhancing neurogenesis and/or neuronalmigration.

[0082] Adult rats and mice were given DiI through intraventricularinjections as described in the example above. At various time intervalsafter the injection (hours-months), the animals were transcardiallyperfused with 15 ml 0.9% saline, followed by 50 ml of +4° C. 4% (w/v)paraformaldehyde and 0.4% (v/v) picric acid in 0.1 M phosphate bufferedsaline, pH 6.9, during 5 min. After brain removal, the tissue was fixedfor an additional 90 minutes in the same fixative and cryoprotected inbuffered sucrose (10% for 24 h, 30% for 2 days) at +4° C. The entiremidbrain was cut with a cryostat using a systematic, uniform randomsampling design, where 40 μm thick frontal sections were taken to sixparallel rostrocaudal series. One series of sections was kept in 0.1 MPBS and the fluorescent signal was immediately converted to a permanentdiaminobenzidine (DAB) signal using a modified previously describedprotocol (Singleton et al., J. Neurosci. Meth. 64:47). Thus, the sampledfreshly cut free-floating sections were immersed for 10 min. in 1% H₂O₂in 0.1 M Tris (pH 8.2) and then washed in buffer alone. Then the tissuewas pre-incubated in the dark for 60 min. at +4° C. with filtered DAB(1.5 mg/ml of Tris buffer pH 8.2), rinsed in Tris buffer and thenmounted on a glass slide and covered with fresh DAB solution, which wasreplaced with fresh solution every 30 min. during the photoconversionprocess. On each sampled slide substantia nigra was identified and thesection was irradiated with ultraviolet light utilizing a 10× objectiveand a rhodamine filter in an epifluorescence microscope (Nikon). Thephotoconversion process was carefully evaluated and when all thefluorescent signal in substantia nigra was visualized with the brown DABproduct, the sections were immunohistochemically labeled with a marker(Vector SG, Vector) for dopamine neurons (tyrosine hydroxylase)utilizing the avidin-biotin-immunoperoxidase system (Vector) (Janson etal., Neuroscience 57:931). The five parallel series of sections wereinstead stored in 30% sucrose in 0.1 M PBS at −20° C. until they wereprocessed for immunohistochemistry and analyzed in a confocal laserscanning microscope utilizing several markers for glia and neurons (withappropriate controls) to determine the neuronal phenotype of the DiIlabeled cells in substantia nigra pars compacta (FIG. 8).

[0083] Quantitative estimates of the total number of TH immunoreactivecell bodies counterstained with cresyl violet (TH/CV+ neurons) as wellas TH immunoreactive cell bodies also containing DiI label were made inthe bilateral SNc. Neuronal counts were determined using coded sectionsand a stereological technique, the optical fractionator (Janson et al.,Neuroscience 57:931). Briefly, the unbiasedly sampled sections inrostrocaudal order were analyzed with a CAST-Grid system (ComputerAssisted Stereological Toolbox, Olympus, Albertslund, Denmark), whichconsists of a video camera on an Olympus BH2 microscope with a motorizedspecimen stage and a microcator to monitor movements in the z-axis(Heidenhain, Traunreut, Germany); both are linked to a PC with GRIDsoftware and a high resolution monitor. After encircling the SNc area ineach sampled section at low-magnification, the analysis was performed athigh magnification (100× oil immersion, numerical aperture 1.4). Thisallowed a clear visualization of individual cells in the denselypopulated encircled area as the focus moved through the tissue, whichwas optically dissected into thin slices and assessed by the microcatorwith a resolution of 0.5 μm. A computerized, uniform, systematic randomsampling of small volumes (extending. 6-9 μm along the thickness of thesection) was carried out; neurons with their nucleoli inside thesampling volume that fulfilled the stereological criteria were countedin a known fraction of the entire nigral volume. As described earlier(Chan et al., J. Pharmacol. Exp. Ther. 280:439), nigral neurons werecounted if they showed both Nissl stained perikarya and THimmunoreactivity within the cell body and/or its dendrites. In theseries of sections where the DiI signal was photoconverted, TH+ neuronscontaining DiI were counted (evaluation at 3,600+). The coefficient oferror for each estimate of the total number of labeled neurons indifferent categories was determined. The obtained counts are independentof any dimensional changes in the tissue during processing such asshrinkage, which was determined along the z-axis. The total number ofnigral dopamine neurons at various time points (a few hours to 60 days)were plotted against time, and from the regression curve (r²=0.97) 175new neurons were found to be generated each week in this brain region,which is around one per cent of the total number of nigral dopamineneurons in the mouse.

[0084] Several migratory streams of DiI labeled cells were characterizedas they reached different parts of substantia nigra pars compacta (notknown or described before, FIG. 9). With the application of a modifiedprotocol for cell counting using a fluorescent microscope, the totalnumber of cells in each of the defined migratory streams is beingdetermined. The interpretation that the DiI labeled neurons were indeednewly generated was confirmed with BrdU labeling in animals receivingchronic administration via drinking water (1 mg/ml, see example above).Furthermore, evidence that the ‘new’ neurons were functional anddeveloped appropriate neuronal processes was supported by the finding ofDiI in some of the TH+ striatal nerve terminals at late, but not atearly survival times.

[0085] The Progeny of Labeled Ependymal Neural Stem Cells IncludeNeurons in Several Brain Regions and of Various Phenotypes

[0086] Utilizing the in vivo fluorescent labeling protocol describedabove, we identified several regions of the brain where DiI labeledcells were identified as neurons. These regions include several parts ofthe hippocampus, including the granular layer of the dentate gyrus (FIG.10), cortical layers and subcortical structures such as the presumablygamma-aminobutyric-acid (GABA)-containing neurons in the subthalamic andsubstantia nigra pars reticulata regions as well as serotoninergicneurons in the raphe brain nuclei.

[0087] Generation of Genetically Modified Stem Cells in vitro

[0088] Genetically engineered stem cells were generated by culturing thestem cells as above, and then transfecting them with expression plasmidsor viral vectors. For each transfection, 4 μg DNA (the method wasestablished using the plasmid CMV-GFP from Clontech, encoding the-greenfluorescent protein (GFP) as a reporter) was added to 200 μl culturemedium (defined above) in a 12×75 mm conical tube (15 ml) and gentlymixed. In a second conical tube 15 μl Lipofectamine reagent was addedinto 200 μl culture medium and vortexed gently. The two solutions werethen combined by adding the second to the first and incubated at roomtemperature 45 min. to allow DNA-liposome complexes to form. Then 1.6 mlculture medium was added to the tube with the DNA-liposome complexes andthis solution was overlaid on the cells (which had the majority of theirmedium carefully removed). Cells were incubated 12 hours and then themedium was replaced with the regular culture medium without DNA. GFPdetection was performed in a fluorescence microscope 48-72 hourspost-transfection. These stem cells could be clonally expanded togenerate spheres of undifferentiated genetically modified stem cells.

[0089] Altered Gene Expression in Stem Cells in vivo

[0090] The feasibility of altering gene expression in stem cells in vivowas done by injecting a replication deficient adenovirus carrying thereporter-gene LacZ under the control of the RSV promoter into thelateral ventricles as described above. X-gal staining (described above)demonstrated expression of a reporter-gene in ependymal stem cells, andthus the feasibility of altering gene expression in stem cells. Stemcells carrying genes driving the expression of nerve growth factor,glial cell-line-derived neurotrophic factor (neuronal survival factor),bc1-2 (a gene which will promote the survival of the stem cells) andnurr1 (which may promote the generation of dopaminergic neurons fromstem cells) are being generated.

[0091] The use of Stem Cells from Transgenic Animals

[0092] We have found that it is possible to culture stem cells fromtransgenic animals. For these studies we have used mice carrying theLacZ gene in their genome (Zambrowicz et al. Proc. Natl. Acad. Sci.USA., 94:3789). These mice express the transgene ubiquitously in alltissues (Zambrowicz et al. Proc. Natl. Acad. Sci. USA., 94:3789). Stemcells from these mice were purified and cultured as above. Strongtransgene expression was revealed by X-gal staining as described above.

[0093] Manipulation of Stem Cell Proliferation and Differentiation byTraumatic Injury

[0094] In adult rats a laminectomy was performed at the mid thoraciclevel to expose the spinal cord, and the dorsal funiculus was cuttransversely with microsurgical scissors, and the lesion wassubsequently extended rostrally by a superficial longitudinal incisionin the dorsal funiculus. In other animals, a hole was drilled in theskull and a needle was inserted into the brain tissue to induce aninjury. In some animals the ependymal cells had been labeled 1-10 daysbefore the injury by a DiI injection as described above.

[0095] Quantification of the proportion of ependymal cells thatproliferate at different time points after an incision in the dorsalfuniculus revealed an almost 50-fold increase 1 day after the injurycompared to uninjured animals (FIG. 5). After the first day, theproliferation gradually declined toward normal within one month (FIG.5). Likewise, ependymal cell proliferation was greatly increased in thewall of the lateral ventricle following brain injury.

[0096] In animals in which the ependymal cells were labeled by a DiIinjection prior to the spinal cord or brain injury, an increasing numberof DiI labeled cells were seen progressively further outside theependymal layer over the first four weeks after the injury (FIG. 6). DiIlabeled cells were abundant in the forming scar tissue within one weekafter the lesion and persisted there for at least one year. Within thescar tissue forming at the injury the vast majority of the DiI-labeledcells showed immunoreactivity to glial fibrillary acidic protein, anastrocyte marker, indicating that the majority of progeny from ependymalcells had differentiated to astrocytes (FIG. 6). However, neuronalmarkers were not found in the DiI labeled cells indicating that thesignals required for neuronal transformation of the stem cells were notpresent in this animal model.

[0097] Chemicals Increasing Neurogenesis in Substantia Nigra ParsCompacta in the Midbrain

[0098] Separate mice were given the1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP, RBI, Natick, MA,USA) (40 mg/kg diluted in physiological saline, sc.). This substance isknown for its selective neurotoxic actions on dopamine neurons in themidbrain causing parkinsonism in humans and experimental animals(Langston et al., Science 219:979, Heikkila et al., Science 224:1451).However, the molecule also has structures in common with compounds knownto act as neuroprotective agents in animal models of Parkinson'sdisease, e.g. nicotine (Janson et al., Neuroscience 57:931).

[0099] In our experiments animals were given MPTP or vehicle and thetime course of changes in the number of DiI+ nigral dopamine cells or inthe staining pattern of the migratory streams of DiI+ cells movingtowards this brain region were analyzed (see above, quantitative methodto study neurogenesis in substantia nigra pars compacta in the midbrainafter labeling of ependymal cells). The treatment led to higher numbersof TH+/DiI+ nigral neurons indicating an increased neurogenesis. Thetotal number of nigral dopamine neurons at various time points (a fewhours to 2 months) were plotted against time, and from the regressioncurve (r²=0.744) the number of new neurons found to be generated eachweek in this brain region was increased with 24% compared to animalstreated with vehicle alone. Also, the migratory streams of DiI+ cells inthe midbrain appeared more pronounced in animals treated with MPTP,which can be analyzed quantitatively using the modified stereologicalmethod described above.

[0100] Transplantation of Ependymal Stem Cells

[0101] Ependymal stem cells from the brain or spinal cord of Rosa26transgenic mice were purified and cultured as above. Spheres ofundifferentiated stem cells from these mice were transplanted to thestriatum of adult rats, by stereotaxic injection of spheres in 15 μl oftheir culture medium (described above). The animals were sacrificed 2days later and the brains sectioned and analyzed for the presence ofLacZ expressing cells deriving from the Rosa26 mice by X-gal staining asdescribed above. The grafted cells were scattered in the tissue close tothe insertion canal. These cells often had several processes (FIG. 7).

[0102] Various publications are cited herein, the disclosures of whichare incorporated by reference in their entireties.

[0103] The present invention is not to be limited in scope by thespecific embodiments described which are intended as singleillustrations of individual aspects of the invention, and functionallyequivalent methods and components are within the scope of the invention.Indeed various modifications of the invention, in addition to thoseshown and described herein will become apparent to those skilled in theart from the foregoing description and accompanying drawings. Suchmodifications are intended to fall within the scope of the appendedclaims.

We claim
 1. A method of isolating ependymal neural CNS stem cells from apost-natal animal, which method comprises the steps of (a) screeningsingle cells obtained by dissociating CNS tissue from said animal forcells exhibiting at least one characteristic of an ependymal neuralstein cell; and (b) recovering the cells that exhibit the characteristicor characteristics screened for in step (a).
 2. A method according toclaim 1, wherein said characteristic of an ependymal neural stem cell isthe expression of a specific cell surface protein.
 3. A method accordingto claim 2, wherein the cell surface protein is the Notch1 receptor. 4.A method according to claim 1, wherein said single cells are screenedfor by use of a previously made labeling in vivo of the ependymal cells.5. A method according to claim 4, wherein said labeling is a dye.
 6. Amethod according to any of claims 1-5, wherein said animal is a human.7. A preparation of isolated ependymal neural CNS stem cells, said cellsexhibiting a purity of at least about 10%.
 8. An isolated ependymalneural CNS stem cell obtained by the method according to any one ofclaims 1-6.
 9. An ependymal neural stem cell that has been geneticallymanipulated.
 10. A method of treating a human or animal who suffers froman injury or disease in the CNS, which method comprises theadministration to said human or animal of a pharmaceutically effectiveamount of ependymal neural stem cells.
 11. A method according to claim11, wherein said disease is Parkinson's disease, Alzheimer's disease,multiple sclerosis, amyotrophic lateral sclerosis, spinal trauma, braintrauma, or mental disorders.
 12. Use of an ependymal neural stem cellfrom a post-natal animal or human to develop in vitro assays.
 13. Apharmaceutical preparation comprising at least one ependymal cell and apharmaceutically acceptable carrier.
 14. Use of a neuronal stem cellfrom a post-natal animal to generate neurons and/or glial cells.
 15. Amethod of measuring neurogenesis for in vivo screening of a substanceregulating migration and/or differentiation of neuronal stem cellscomprising labeling neuronal stem cells in an animal and administeringto the animal the substance and assaying the migration ordifferentiation of the stem cells.
 16. A substance obtained by thescreening according to claim
 15. 17. An animal model for use in themethod according to claim 15.